High throughput amplicon sequencing of genomic regions of interest can be very useful for a variety of molecular genetic genotyping applications, including forensic and clinical sample analysis. Some conventional sequencing platforms provide for rapid, high yield sequence data, which can enable the sequencing of multiple amplicons from many samples in a short period of time. However, efficient use of at least some of these sequencing platforms requires the use of sample barcoding, which can be cumbersome and expensive when dealing with tens to thousands of samples. This complication is further compounded if multiple amplicons are to be sequenced from each sample. As such, there is a demonstrated need to develop systems and methods that enable the production of sequencing platform-ready amplicons in a multiplex fashion with a common set of indexing oligonucleotide sequences that can be used in preparing any amplicon for sequencing.
For example, PCR reactions can be used to generate amplicons that are sequencing-platform ready for some systems (e.g., bacterial ribosomal gene sequencing) (J G Caporaso et al. (2012) ISME J. 6(8) 1621-1624). Moreover, some conventional methodologies include indexing, which comprises the use of a plasmid for production of the indexing oligonucleotide (Chubiz et al. (2012) PLoS One 7(10) e47959). Other conventional sequencing platforms may be compatible with custom amplicon sequencing products designed for different eukaryotic organisms, but these conventional systems utilize an initial hybridization of probes with an extension-ligation reaction followed by a PCR reaction requiring at least 50 nanograms (ng) of pure, high quality DNA template as starting material. As such, there is a demonstrated need for systems and methods that provide for substantially or completely simultaneous amplification of multiple amplicon targets from any organism in a PCR reaction with subsequent universal indexing addition that requires only a single set of common indexing oligonucleotides that can be used with any set of amplicons from any type of nucleic acid-based sample, regardless of the quality of the sample.